There is currently a need in drug discovery and development and in general biological research for methods and apparatus for accurately performing cell-based assays. Cell-based assays are advantageously employed for assessing the biological activity of chemical compounds.
In addition, there is a need to quickly and inexpensively screen large numbers of chemical compounds. This need has arisen in the pharmaceutical industry where it is common to test chemical compounds for activity against a variety of biochemical targets, for example, receptors, enzymes and nucleic acids. These chemical compounds are collected in large libraries, sometimes exceeding one million distinct compounds.
Performing cell-based assays often involves recording cellular images and quantifying these images using algorithms of image analysis software. Instruments are known for imaging fluorescently labelled cells and software of these instruments have a number of analysis modules which quantify, for example, biological protein translocations and reactions to stimuli within the fluorescently labelled cells.
Analysing such cellular images involves identifying regions of the image corresponding to the cells and this is generally achieved by detecting fluorescent radiation emitted by a fluorescently labelled cell. One example of such a label is a marker stain which labels the cell nucleus, for example HOECHST 33342™ manufactured by Invitrogen, or DRAQ5™ manufactured by Biostatus.
In order to label the cell nucleus, these nuclear stains interact with the nucleic acid structure of the nucleus. This interaction, over a period of time, proves to be toxic to the cell. It is important that data collected when performing cell-based assays is as accurate as possible and the toxicity of nuclear stains therefore prevents their use in live cell assays where it is desired to mark and measure cell responses at intervals over a relatively long time period.
Marking cell organelles other than the nucleus is often difficult. Markers for the cytosol, plasma membrane or Golgi apparatus are not effective over a relatively long period of time as they degrade or relocalise within the cell over time.
The use of contrast agents in imaging of biological objects is known in the art.
U.S. Pat. No. 6,187,289 discloses the use of acetic acid as a contrast agent in reflectance confocal imaging of cells.
Methods employing contrast-coloring dyes in combination with precipitable dyes have been reported in U.S. Pat. No. 6,090,541 to enhance the visualization of bacteriophage-derived plaques in confluent lawns of bacteria. The contrast-coloring dyes are capable of coloring the support material while the precipitable dyes serve as a substrate for a bacterial or viral enzyme to produce a colored precipitate.
Metal complex contrast agents are known for MRI and X-ray imaging, as disclosed in WO 01/17567.
It is an object of the invention to provide an improved method of imaging biological objects during a biological assay.